Phylogenetic groups, extended-spectrum β-lactamases and metallo-β-lactamase in Escherichia coli isolated from fecal samples of patients with diarrhea in Iran.

AIM
The aims of this study were to investigate the phenotypic and genotypic of extended-spectrum β-lactamase (ESBL) and metallo-β-lactamases (MBLs) and determine phylogenetic background E. coli isolates from fecal samples of patients with diarrhea in Kerman, southeast of Iran.


BACKGROUND
The emergence of ESBLs and MBLs-producing E. coli caused problems in antibiotic treatments. E. coli strains can be assigned to four main phylog-groups, including: A, B1, B2 and D.


PATIENTS AND METHODS
E. coli isolates (n=216) were obtained from fecal samples of patients with diarrhea between June and December 2013. ESBLs and MBLs were confirmed by disk-diffusion and broth micro-dilution methods. Using PCR, the ESBL-positive isolates were screened to determine the phylo-groups and the presence of bla CTX-M-15, bla OXA-1, bla PER-1, bla VIM and bla IMP genes.


RESULTS
ESBL-positive isolates (n= 56) were detected. Among ESBL-positive isolates, 51 isolates were positive for bla CTX-M15 and one isolate was positive for both bla CTX-M-15 and bla OXA-1 genes. None of the isolates were positive for bla PER-1, bla VIM and bla IMP genes. PCR assay for phylotyping of isolates indicated that the isolates were belonged to groups A (54.16%), B1 (11.11%), B2 (12.96%) and D (21.75%). The isolates possessed bla CTX-M-15 gene were belonged to A (35 isolates), B1 (5), B2 (3) and D (8) phylo-groups.


CONCLUSION
Our results indicate that bla CTX-M-15 gene is widespread among diarrheagenic E. coli isolates. ESBL-producing E. coli isolates were disseminated among a diversity of phylo-groups. Further studies are necessary to identify the ESBL genes in relation to phylogenetic groups.


Introduction
1 Diarrheal diseases are one of the important causes of morbidity and mortality in developing countries. been one of the most significant epidemiologic changes in infectious disease (4). Types of enzymes conferring resistance to β-lactam antibiotics have emerged due to antibiotic selection pressure; most alarming are the ESBLs produced by enteric pathogens that have spread throughout the world (5). ESBLs confer resistance to antibiotics including, third and fourth-generation cephalosporins and monobactams. CTX-M enzyme types have emerged worldwide among Enterobacteriaceae species, in particular E. coli. The CTX-M-1 β-lactamase is the most prevalent classes of enzymes throughout the world with the CTX-M-15 being the most recognized variants. CTX-M-14 and CTX-M-15 clusters are widely diffused among humans in Europe (3). During the last four years descriptions of new OXA-type β-lactamases have increased considerably. Thus, Poirel et al. reported in 2010 that 147 OXA enzymes had been assigned to this group. Today this number has reached 227 OXA-type betalactamases (6). OXA-type β-lactamases have been detected in many Gram-negative bacteria, in particular Pseudomonas aeruginosa. However, among OXA-type β-lactamase, OXA-1 has been found in 1-10% of E. coli isolates (7). PER-1 is widely distributed in Turkey and share 86% amino acid homology with PER-2, which is found mostly in South America, several countries in Europe and the Far East (8). Metallo-β-lactamases (MBLs) are founded increasingly in Gram-negative organisms and are recognized mostly in Pseudomonas species. MBLs hydrolyze all β-lactams, including carbapenems. Among Enterobacteriaceae species, the bla IMP and bla VIM genes have been identified throughout the world (9). The E. coli species are genetically diverse, and strains have been divided into four major phylogenetic groups, the most frequent ones being A, B1, B2, and D. The most diarrheagenic E. coli strains belong to group D and the commensal strains to groups A and B1. However, ESBL-producing E.
coli is genetically various (10). The purposes of the current study were to determine the phylogenetic groups and assess phenotypic and genotypic of ESBL and MBL-producing E. coli isolates from fecal samples of patients with diarrhea in Kerman, southeast of Iran.

Bacterial isolates
The research study was approved by the department of Microbiology, International Branch, Shahid Beheshti University of Medical science, Tehran, Iran. E. coli isolates (n=216) were obtained from patients with diarrhea who referred to different clinical laboratories and hospitals in Kerman city (southeastern of Iran) between June to December 2013. Diarrheic samples were identified as the incidence of watery and unformed stools with or without one of the following symptoms: fever, nausea, abdominal cramps, and tenesmus. Their age ranged from <5 years old (69), 5 to 15 years old (44), 15 to 40 years old (64) and 40 to 80 years old (39). Stool specimens were directly streaked onto Mac-Conkey agar (Biolife Laboratories, Milan, Italy) for isolation of E. coli. After overnight incubation at 37°C, lactose-fermenting colonies were selected and identified by the biochemical and bacteriological tests.

PCR
Whole-cell DNA was extracted using the lysis method. ESBL-positive isolates were screened by PCR for the carbapenemase-encoding genes, bla IMP and bla VIM (11). Detection of other β-lactamase genes such as bla CTX-M-15 , bla OXA-1 and bla PER-1 was performed with amplification conditions, as described previously (12)(13)(14). All E. coli isolates were assigned to phylogenetic groups using the multiplex PCR method (15). On the basis of presence or absence of the chuA, yjaA genes and an anonymous DNA fragment, TspE4.C2 by multiplex PCR, the E. coli isolates were assigned to one of four groups (A, B1, B2 and D). The specific primers used for detecting sequences encoding MBLs, ESBLs and phylogenetic groups are presented in Table 1.

Modified Hodge test
Phenotypic confirmatory test for carbapenemase production in E. coli isolates was done by modified Hodge test using meropenem (10 ug) as the indicator disc according to CLSI guidelines 2013 (16).

Results
From the 216 E. coli isolates, 56 (25.92%) ESBL-positive isolates were identified by confirmatory tests (double-disc synergy test (DDST) as a standard disc-diffusion assay and broth micro-dilution) according to CLSI 2013.
Among ESBL-positive isolates, 51 isolates (91.07%) were positive for bla CTX-M-15 and one isolate (1.78%) was positive for both bla CTX-M-15 and bla OXA-1 genes (Figure 1 and 2). The survey of ESBL-positive isolates indicated that none of the isolates were positive for bla PER-1 , bla VIM and bla IMP genes.
The minimum inhibitory concentration of 88 E.
coli isolates resistance against at least one of the antimicrobial drug by disc-diffusion method was done to five antibiotics. Base on the guidelines of CLSI 2013, the isolates of E. coli tested were resistant to cefotaxime (61 isolates), ceftazidime (38), cefepime (29) and imipenem (2) All strains were sensitive to meropenem.

Discussion
Extended-spectrum-β-lactamase and metallo-βlactamases are reported increasingly in Gramnegative organisms. Unfortunately, extensive use of antibiotics is the cause of resistance phenomena, and treatment of various infections faces a serious problem (9,17).
In this study, the prevalence of ESBL-positive  (23,24). In developing countries, most patients received antibiotics treatment without prescription; such common practices in nearly all developing countries exert a selective pressure on E. coli, whereas in more developed countries effective strategies for the control of antimicrobials are present, which effectively prevents the emergence of ESBLs (4).
Several worldwide reports have shown the emergence of bla CTX-M producing E. coli as a significant pathogen since the turn of the 21 st century (25) coli (stool, urine, soft tissue, exudates and blood specimens), revealed that 61.8% of the 144 CTX resistant E. coli isolates carried the bla CTX-M-1 group alleles. None of the isolates were positive for CTX-M groups 2 and 9 (18). McGettigan et al., also reported that 48% of cephalosporinresistant E. coli isolates produced CTX-M-type ESBLs in Philadelphia (27). In Iran, the frequency of ESBL positive isolates of Enteroaggregative E. coli in children was 52.7%. The frequency of bla CTX-M in these isolates was 63.1%. The results of this study indicate that one E. coli isolate carries bla CTX-M-15 and bla OXA-1 genes, which is similar to the results of the Peirano et al. (25).
PER type β-lactamases have a high prevalence in Turkey and it's possible that the dissemination of them in Western Europe and the Far East is mostly related to the widespread immigration of Turkish nationals (29). Similar to previous studies, we couldn't find any bla PER among the E. coli isolates in Iran and Thailand (29,30).
Metallo-β-lactamases have been reported from many countries, particularly in Gram-negative organisms like P. aeruginosa and Acinetobacter species. None of MBL producing E. coli was detected in this study. In their study, Khoshvaght et al. has evaluated the presence of bla IMP , bla VIM and bla NDM-1 genes from EAEC isolates and founded that none of the isolates possessed these genes (29).
Phylogenetic analyses in the current study showed that E. coli isolates fall into four main phylogenetic groups (A, B1, B2, and D), which most diarrhea isolates belong to A and D groups.
In a study from southeast of Iran, diarrheic E. coli isolates mostly fell into phylogenetic groups A and D (31). Several studies indicated that diarrheagenic E. coli strains belong to groups A, B1 and D (10,15). Tropical populations strains associated with severe diarrhea seem to preferentially harbor strains of phylogenetic group A and to a lesser extent B1, these strains might have the necessary genetic background for the emergence of pathogenic intestinal strains. This might be one of the agents explaining the higher prevalence of diarrhea in tropical areas (32).
One of the purposes of this study was to determine to ESBLs and MBLs genes in relation to phylogenetic background in E. coli isolates from diarrheal patients in southeast of Iran. coli isolates in Spain (35). A study on ESBLproducing E. coli isolates recovered from blood between 2000 to 2010 of Canadian cases indicated that the majority of isolates belonged to phylogenetic groups (B2, D, A and B1) (25). The presence of bla CTX-M-15 gene in all major phylogenetic groups reflects the widespread of these enzymes across the diversity of E. coli strains. However, findings of the current study indicated that most group B2 and B1 phylotypes were devoid of bla CTX-M-15 gene. The hypothesis might explain this observation, which group B2 might be less exposed to antibiotics than groups A, B1, and D. The phylogenetic group B2 strains mostly carry more virulence genes than other phylo-types and mostly belonged to extraintestinal pathogenic E. coli, whereas other phylo-groups are predominant in the fecal E. coli population of healthy subjects (10). Clermont et al, reported that CTX-M-15-producing E. coli is associated with a high level of antibiotic resistance and virulence genes that belonged to B2 phylo-group, showing that under certain selective pressures, the previously observed trade-off between resistance and virulence genes may not apply (36).
In the current study, imipenem was found to be the most active agent, while resistance to cefotaxime was high. Although resistance to cefotaxime is reported to vary in different areas, and imipenem resistance is still very rare around the world including Iran (17).
According to the broth micro-dilution results, two isolates were resistant to imipenem; however, results of modified Hodge method showed that none of the isolates produce carbapenemase enzyme The modified Hodge test was weakly positive for carbapenemase production by the E. coli isolate and strongly positive for carbapenemase production by the Klebsiella pneumoniae isolate (37).
The results of this study indicate that some ESBL coding genes are widespread among diarrheic isolates. The positive isolates for bla  gene are usually distributed among phylogenetic groups other than B2 and B1 phylogroups, whereas ESBL-producing E. coli isolates were disseminated among various phylogenetic groups. Further studies and detailed analysis of the data are necessary to detect the ESBL genes in relation to phylogenetic background.